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Release of inflammatory mediators by human basophils

Lie, W.J.

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Citation for published version (APA):Lie, W. J. (1999). Release of inflammatory mediators by human basophils.

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Download date: 31 May 2020

Chapter 2

Degranulation of human basophils by picomolar concentrations of lnterleukin-3, Interleukin 5 or Granulocyte-

Macrophage Colony-Stimulating factor

W. Johan Lie, Frederik P.J. Mul, Dirk Roos Arhur J. Verhoeven and Edward F. Knol

m ;

Chapter 2

Abstract

In most secretory cells, an increase in the cytosolic free Ca2* concentration ([Ca2+]i) is associated with the exocytosis response. In the present study, we have evaluated the effect of thapsigargin on histamine release from purified (70 to 97% pure) human basophils of non-allergic donors. Thapsigargin (2 uM), by inhibiting the uptake of Ca2+ in the stores of the endoplasmic reticulum, leads within 1 minute to a gradual increase in [Ca2*]: in human basophils. Incubation of basophils with thapsigargin by itself induced only a very small release of histamine (5.6 ± 1.8%). However, under suboptimal conditions of stimulation with other agonists, pre-incubation of basophils with thapsigargin significantly enhanced histamine release. Most strikingly, thapsigargin addition made basophils extremely sensitive for histamine release induced by interleukin-3 (IL-3; max histamine release 71 ± 7%), IL-5 (43 ± 8%) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 57 ± 10%). These cytokines by themselves did not induce histamine release in purified basophils. The effect of thapsigargin was mimicked to a limited extent by addition of platelet-activating factor (PAF). We conclude that depletion of the Ca2* stores may be a critical event in the activation of receptor-mediated histamine release in human basophils.

40

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

Introduction

Basophilic granulocytes can be found in the lungs of allergic asthmatics1"3, in nasal mucosa and nasal secretions of allergic rhinitis patients4,5, and in skin lesions of atopic dermatitis patients57. The histamine detected during the late phase of the allergic response is thought to be released from basophils in these

6,8,9

tissues The mechanisms controlling histamine release by basophils have been

shown to involve second messengers specifically induced by different stimuli. For instance, cross-linking of IgE on the basophil membrane by anti-lgE induces histamine release via activation of PI 3-kinase (as shown by the potent inhibition by wortmannin at nanomolar concentrations) and not via a pertussis toxin-sensitive G-protein10,11. Whereas fMLP-induced histamine release is independent of PI 3-kinase, it is strongly inhibited by the addition of pertussis toxin10,11. An increase in the cytosolic free Ca2+ concentration [Ca2+]; accompanies the degranulation induced by anti-lgE or fMLP addition2,13. However, further studies have revealed that this relation is not strict, because incubation of basophils with PMA results in degranulation without a rise in [Ca2*]] 12. On the other hand, some of the chemokines, such as IL-8, do induce a rise in [Ca2+]i, but do not induce histamine release from basophils14.

A rise in [Ca2+]j in cells without activation of cell membrane receptors can be achieved by inhibitors of sarco(endo)plasmic reticulum Ca2+-ATPases, such as thapsigargin15. Thapsigargin depletes the same intracellular Ca2+ pool that is depleted by receptor agonists, without increasing the intracellular concentration of inositol phosphates16. In several cell types it has been shown that depletion of the intracellular Ca2+ stores can open plasma membrane Ca2+ channels17. It has been suggested that a soluble mediator is released from the depleted Ca2+

stores that triggers Ca2+ influx18. Another model suggests a direct contact between the stores and the plasma membrane mediating store-operated Ca +-influx19. Whatever mechanism is involved, we have analyzed the role of [Ca2+]j changes in human basophils following depletion of intracellular Ca2+ stores by thapsigargin in combination with several agonists. In this way it became clear that thapsigargin can complement the action of stimuli that do not change [Ca2*]! by themselves.

Material and Methods

Materials Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden), human albumin (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service

41

Chapter 2

(CLB), Amsterdam, The Netherlands), sheep serum containing IgG antibodies against human IgE (CLB, no SH25P01), monoclonal antibodies CD2 (CLB, CLB-T11), CD14 (CLB, CLB-mon/1), CD16 (CLB, CLB-gran/1), CD19 (CLB, CLB-B4) and CD66b (CLB, CLB-gran/10) and recombinant IL-3 (Boehringer Mannheim, Mannheim, Germany) were obtained from the manufacturers. The concentration of anti-lgE antibodies in the sheep serum was calculated by comparing affinity-purified sheep anti-human-lgE antibodies with serum dilutions for histamine release from human basophils. Recombinant IL-5 and recombinant GM-CSF were a kind gift of Glaxo (Greenford, UK) and Sandoz Pharma (Uden, The Netherlands), respectively. Goat-anti-mouse-lgG-coated dynabeads were obtained from Dynal A.S.(Oslo, Norway). HEPES was obtained from Sigma Chemicals (St. Louis, MO, USA). Thapsigargin (Sigma Chemicals), 2,5-di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (a kind gift of Dr. K-H. Krause, Geneva, Switzerland), cyclopiazonic acid (CPA) (Sigma Chemicals), PMA (Sigma Chemicals), FMLP (Sigma Chemicals), A23187 (Sigma Chemicals) and lndo-1/AM (Molecular Probes, Junction City, OR, USA) were dissolved in DMSO and incubation media. The final concentration of DMSO (< 0.3%, v/v) had no effect on cell viability or histamine release.

Purification of basophils Buffy coats were prepared from 500 ml of human blood after informed consent had been obtained from healthy donors without an allergic history. The basophils were purified by successive isopycnic centrifugation and elutriator centrifugation, as previously described by De Boer and Roos20 (purity 75% ± 10%). The cells were resuspended in incubation medium (132 mM NaCI, 6 mM KCl, 1 mM MgS04, 1.2 mM potassium phosphate, 20 mM HEPES, 5.5 mM glucose and 0.5% (w/v) human albumin, pH 7.4). For determination of changes in cytosolic free Ca2+ ([Ca2+]i), the basophils were further purified by immuno-depletion of residual contaminating cells with various monoclonal antibodies (CD2, CD14, CD16, CD19, CD66b) and magnetic beads21. The cells were counted electronically (Coulter counter model ZF; Coulter Electronics, Dunstable, UK). The percentage of basophils was determined by differential staining with Alcian blue22 and from cytocentrifuge preparations stained with May-Grünwald/Giemsa. Viability was determined by trypan blue exclusion. The purity of the basophils in these preparations was > 95%, with a viability of about 98%.

Basophil loading with indo-1 The cells were centrifuged once, were resuspended at a concentration of 107

cell/ml in incubation buffer supplemented with 1 mM CaCb and were loaded with indo-1 by incubation with 1 uM of the acetoxy-methyl ester indo-1/AM for 30

42

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

min at 37°C. The cells were then washed and resuspended in incubation medium to a concentration of 107 cells/ml.

Measurement of [Ca2*], in basophils in suspension Fluorescence measurements were performed in a spectrofluorophotometer (model RF-540; Shimadzu, Kyoto, Japan). The contents of the cuvette were continuously stirred and the cuvette holder was kept at 37°C. The excitation and emission wavelengths were taken at 340 and 390 nm, respectively. Before each measurement, the cells were diluted in incubation medium to a concentration of 7.5x105 cells/ml and were incubated for 5 min at 37°C. The medium was supplemented with 1 mM CaCI2 unless indicated otherwise. Calibration was performed by permeabilization of the cells afterwards with 5 mM digitonin to obtain maximal fluorescence and then quenching the signal with 0.5 mM MnCb to obtain minimal fluorescence23. The cytoplasmic free calcium concentration was calculated by the equation: [Ca2+]j = Kd x ((F-Fmin)/(Fmax-F))24, using 250 nM as the Kd for the Ca2+/indo-1 complex25. Leakage of indo-1 from the basophils, as previously observed with basophils of rat origirP6, did not occur, as indicated by the absence of fluorescence changes after addition of Mn2+ to intact human basophils. Furthermore, uptake of indo-1 in basophil granules, which might cause an artefactual increase in fluorescence upon degranulation27

did not occur, as indicated by the absence of fluorescence changes upon addition of the secretagogue PMA.

Measurement of [Ca2*], in basophils in suspension at the single cell level The flow cytometric analysis of the indo-1-loaded basophils was performed with a FACSTAR-PLUS (Becton Dickinson, San Jose, CA, USA) equipped with a sample delivery system. Indo-1 was excitated by 100 mWatt UV (351-364 nm) illumination from an argon ion laser (Innova 90/5, Coherent, Palo Alto, CA, USA). Free indo-1 emission was detected at 463-507 nm (DF 470/22 nm) and Ca2+/indo-1 emission at 385-425 nm (DF 405/20 nm). Both emission signals were separated by means of a 505 nm short pass filter placed under an angle of 45°, and were linearly amplified. The cells were continuously analyzed at a rate of about 200 per second. Before each measurement the basophils were diluted 30-fold to a concentration of 3x105/ml in incubation medium with 1 mM CaCb present, and incubated at 37°C with magnetic stirring. Stimulation took place without disturbing the sample flow. Forward and sideward scatter patterns were used to gate out contaminating neutrophils, eosinophils and monocytes. Thus, an almost pure basophil population was analyzed, with < 3% lymphocytes present. The ratio 405/470 nm was calculated with the kinetic software INCA28.

43

Chapter 2

Histamine release

Purified basophils were washed and resuspended in incubation medium supplemented with 1 mM CaCI2. Incubations were performed with 105 cells in 450 pi. Before reagents were added, the cells were prewarmed for 5 min at 37°C. After stimulation for various times, the cells were pelleted for 20 sec at 12000 x g, and 350 pi of supernatant was collected. The supernatant was mixed with 200 pi of 12% (w/v) perchloric acid and 750 pi of saline (0.9% w/v), and stored at 4°C. For measurement of the total amount of histamine, 105 cells were lysed in perchloric acid. Histamine was measured by fluorometric analysis as described by Siraganian29. Histamine release was calculated as percentage of the total amount of histamine in the cells. Spontaneous histamine release was not subtracted from the histamine release observed.

Statistics

All data are presented as mean ± SEM. Statistical significance of differences were calculated by the Student's t-test.

Results

Thapsigargin-induced changes in [Ca2+l in human basophils It has been demonstrated in several cell types that addition of thapsigargin results in blocking of the microsomal Ca2+-ATPase, resulting in a gradual

,2+ increase in [Ca ], due to uncompensated leakage from Ca2+ stores30. We have analyzed the effect of thapsigargin on purified basophils after loading the cells with the Ca2+ indicator indo-1. As shown in Figure 1, addition of thapsigargin (2 pM) resulted in a gradual and sustained increase in [Ca2+]i from 60 nM to about 250 nM. The same effect, but less pronounced and at higher inhibitor concentrations (50 pM), was found with two other blockers of the Ca2+-ATPase, cyclopiazonic acid (CPA) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (DBHQ)31

(Figure 1).

50

Thap

CPA

DBHQ

stimulus

Fig 1. Increase in [Ca2+]; in basophils after addition of thapsigargin (2 pM), CPA (50 pM) or DBHQ (50 pM) measured in suspension. Basophils were loaded with indo-1 and incubated at 37°C as described in Materials and Methods. After 5 min the stimuli were added. A representative graph is shown of 4 independent experiments.

44

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

To investigate whether the [Ca2+]; increase by thapsigargin was also due to Ca2+

influx we studied the influence of thapsigargin in medium without extra Ca2+

added. As shown in Figure 2 the [Ca2+]j increase is due to release from the stores as well as due to influx.

250

100

50 -

time 1 min

Fig 2. Increase in [Ca2+]; in basophils after addition of thapsigargin (2 uM), measured in suspension with (a) and without (b) 1 mM of CaCI2 added. Basophils were loaded with indo-1 and incubated at 37°C as described in Materials and Methods. After 5 min thapsigargin was added. After 5 min, in trace b. 1 mM of CaCI2 was added. A representative graph is shown of 3 independent experiments.

One of the intriguing properties of human basophils is that after in vitro activation with for instance anti-lgE or fMLP, degranulation occurs in some but not in all cells, following an all-or-nothing-like response32. As shown in Figure 3 thapsigargin induced a rise in [Ca2+]: in almost all cells. This effect was reproduced by the two other blockers of the Ca2+-ATPase, CPA and DBHQ (Figure 3).

The effect of thapsigargin on histamine release Although thapsigargin induced a marked increase in [Ca 2 ! , it only induced a slight degranulation of human basophils (Table I). The rapid induction of histamine release by fMLP or C5a, at optimal concentrations of those stimuli (1 pM fMLP or 10 nM C5a), was unaffected (fMLP), or slightly inhibited (C5a), by thapsigargin. However, in the presence of suboptimal concentrations of fMLP (10 nM) or C5a (0.1 nM), thapsigargin strongly potentiated basophil degranulation to values reached with optimal concentrations for these stimuli. Thapsigargin strongly enhanced two other major pathways of basophil stimulation10, i.e. those induced by anti-lgE and by PMA. The most pronounced

45

Chapter 2

Thap CPA DHBQ

100 200 300 400 0 100 200 300 400 100 200 300 400

time (sec)

Fig 3. Increase in [Ca2+],in basophils after addition of thapsigargin (2 pM), CPA (50 |JM) or DBHQ (50 pM) measured at the single-cell level. Basophils were loaded with indo-1, incubated at 37°C and processed for analysis in a flow cytometer as described in Materials and Methods. The figure shows the change in fluorescence ratio of 405/470 nm induced by thapsigargin in the presence of 1 mM CaCI2 The horizontal white line represents mean of resting value. A representative result of 3 independent experiments is shown.

effect of thapsigargin was found on PMA-induced degranulation measured after 30 min (Table I).

Table I: Effect of thapsigargin on histamine release from human basophils

Condition Control Thapsigargin Control 2.6 + 0.4 C5a, 0.1 nM 7.1 ±2.5 C5a, 10 nM 61 ±11 fMLP, 10 nM 13 ± 3 fMLP, 1 uM 39 + 7 anti-lgE, 3 ng/ml 6.7 ±4.2 anti-lgE, 100 ng/ml 35 ±8 PMA, 100 ng/ml (30 min) 20 + 5 PMA, 100 ng/ml (1 hr) 50 ±12

5.6 ±1.8* 40 ±20 43 ±9* 34 ± 7** 38 ± 6 17 ± 11 55 ± 9** 83 ± 7 " 82 ± 4**

Basophils were incubated for 5 min at 37°C with 2 uM thapsigargin or with solvent (control), before the indicated stimuli were added. After 45 min (unless indicated otherwise) of histamine release was determined in the cell-free supernatant. Data are expressed as % of histamine released (mean ± SEM of 3 or more experiments). *P<0.05; "PO.02.

46

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

Cytokine-induced histamine release after thapsigargin pretreatment Previously, we have demonstrated that addition of IL-3 results in homotypic aggregation of basophils, but does not induce degranulation of purified human basophils obtained from non-allergic donors32. Clearly, IL-3 induces an early signal in human basophils without affecting [Ca2+]j or inducing a rapid release response12,33. We therefore hypothesized that the increase in [Ca2+]i caused by depletion of Ca2+ stores by thapsigargin might render the basophils susceptible for histamine release upon addition of IL-3. We included in these experiments the effect of thapsigargin on the kinetics of the PMA-induced histamine release, because PMA also induces early signals in basophils before histamine release is apparent10,32. As shown in Figure 4, addition of IL-3 to thapsigargin-treated basophils resulted in a marked histamine release, whereas no histamine release occurred without thapsigargin pretreatment.

100 y

0) 10 CO CD

C

'E to to

-•- thap/IL-3

-•- thap/PMA -O- IL-3 -?- PMA

10 20 30

time (min)

40 50 60

Fig 4. Time course of cytokine or PMA-induced histamine release from basophils after thapsigargin pretreatment. After 5 min preincubation at 37°C, the basophils were incubated with 2 uM thapsigargin (closed symbols), or solvent (open symbols) for 5 min. The basophils were then stimulated with 500 pM IL-3 (circles) or 100 ng/ml PMA (triangles). After incubation for various times, histamine release was stopped by diluting the cells in ice-cold PBS. Results are the mean ± SEM of 3 experiments.

The same effect, albeit to a lower extent, was achieved by the other Ca2+-ATPase inhibitors CPA and DBHQ (Table II). The histamine release induced by PMA in thapsigargin-treated cells was much more rapid than by PMA alone. The response after IL-3 addition to thapsigargin-treated cells was significantly slower than after PMA stimulation of thapsigargin-treated cells (Figure 4). It was remarkable that an increase in [Ca2+]i induced by A23187 did not lead to histamine release by IL-3, nor did it affect the histamine released by PMA.

Thapsigargin not only potentiated the effect of IL-3 on basophil degranulation, but also that of IL-5 and GM-CSF (Table III). The potency of IL-3 to induce histamine release under these conditions was higher than that of the other cytokines. Therefore, we studied the effect of different concentrations of

47

Chapter 2

thapsigargin in combination with IL-3. Figure 5 shows that doses near 2 uM of thapsigargin in combination with IL-3 are optimal for histamine release.

Table II: Effect of [Ca +], elevation on histamine release from human basophils

Stimulus Pretreatment none IL-3 PMA None 2.3 ±0.5 3.9 ± 1.2 6.3 ±1.0* Thapsigargin (2 uM) 5.5±1.6 42 ±10** 76 ±7* CPA (50 uM) 3.1 ±0.5 23 ± 8*** 60 ±10* DBHQ (50 uM) 2.4 ±0.1 17 ±5*** 49± 11* A23187(1 uM) 2.7 ±0.3 5.3 ±2.7 4.2 ±0.6

Histamine release was determined after an incubation of the basophils for 5 minutes at 37°C with the agents indicated before IL-3 (600 pM) or PMA (100 ng/ml) was added. After another 15 minutes of incubation, samples were taken for histamine release in the cell-free supernatant. Data are expressed as percentage of histamine released (mean ± SEM of three experiments). Probability (p) values are for IL-3 or PMA-stimulated cells versus untreated cells or cells treated with Ca2+-ATPase inhibitors only. ***p < 0.005, **p < 0.02, *p < 0.01

Table III: Histamine release from thapsigargin-treated basophils after cytokine addition

Cytokine ED50 (pM) Max.release (% of total) IL-3 IL-5 GM-CSF

6 ± 2 41 ± 4 140 ± 2 5

71 ± 7*** 43 ± 8*** 57 ±10*"

Basophils were incubated for 5 min at 37°C with 2 uM thapsigargin before different concentrations of the indicated cytokines were added. After 45 min, histamine release was determined in the cell-free supernatant. The dose necessary to achieve a half-maximal response (ED50) was calculated. Histamine release with cytokines alone (up to 10 nM) never exceeded 5% of total histamine content. Data are expressed as % of histamine released (mean ± SEM of 3 or more experiments). ***: P<0.01

PAF-induced histamine release after IL-3 pretreatment We next sought for a physiological agent that, like thapsigargin, results in Cef+

store depletion without inducing much histamine release by itself. Previously, it has been found that IL-3 renders basophils susceptible for a subsequent stimulation by the inflammatory mediators IL-8 or platelet-activating factor (PAF), resulting in histamine release34,35.We found that PAF increased [Ca2+]i in human basophils in a transient fashion, (Figure 6) but that it did not induce

48

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

0

(0

E ra

75

50

25

-O no IL-3 -•- IL-3,600 pM

1000 10000

thapsigargin (nM)

Fig 5. Histamine release from basophils after thapsigargin pretreatment. After 5 min preincubation at 37°C, the basophils were incubated with different concentrations of thapsigargin. The basophils were not stimulated any further (open circles), or stimulated with 600 pM IL-3 (closed circles). After incubation for 25 min, histamine release was stopped by diluting the cells in ice-cold PBS. Results are the mean ± SEM of 3 experiments.

4 -

- 1

Fig 6. Increase in [Ca2+]i in a basophil suspension after addition of PAF (1 uM) measured at the single-cell level. Basophils were loaded with indo-1, incubated at 37°C and processed for analysis in a flow cytometer as described in Materials and Methods. The figure shows the change in fluorescence ratio of 405/470 nm induced by PAF in the presence of 1 mM CaCI2. A representative result of 3 independent experiments is shown.

t PAF (1 pM)

0 50 100 150 200 250 Time (sec)

histamine release from purified basophils obtained from healthy individuals. However, treatment with PAF did permit IL-3-induced histamine release, although the response was much lower than that found with thapsigargin and IL-3 (Figure 7). In these experiments the order of stimulus addition (see legend of Figure 7) proved to be of major importance. When IL-3 was given during or after PAF-induced elevation of [Ca2+]i , the cytokine was not able to induce a significant histamine release (Figure 7). This time-dependence was not seen with thapsigargin.

49

Chapter 2

e 'E to «-» CO !c >< ca E

125 y

1 0 0 -

75-

50

•ft 25

stimulus

PAF Thapsigargin

X I

X i

Fig 7. Effect of timing of additions on IL-3-induced histamine release in PAF-treated or thapsigargin-treated cells. After 5 min preincubation at 37°C, the basophils were treated with PAF (1 uM) or with thapsigargin (2 uM). In addition, the basophils were stimulated with IL-3 (250 pM) at various times before or after PAF or thapsigargin addition as indicated in the figure. Basophils were incubated for 30 min after the last addition had been made. Histamine release was determined in the cell-free supernatant. Results are expressed as percentage of histamine released

after PAF addition with 1 min IL-3 preincubation (amounting to 23.6 ± 7.5%) or after thapsigargin addition with 1 min IL-3 preincubation (amounting to 68 ± 12.2%) (mean ± SEM of 3 experiments).

-10 -1 1 10 -10 -1 1 10

timepoint of IL-3 addition

Discussion

The results of our study on the effects of thapsigargin on basophil degranulation has two important implications. First, the cytokines IL-3, IL-5 and GM-CSF exert a priming effect on basophil histamine release that awaits a complementary signal, probably generated as a consequence of prolonged depletion of intracellular Ca2+ stores, before degranulation can occur. Second, the role of changes in [Ca2+]i should be taken into account when studying the degranulation mechanism of basophils.

The most likely explanation for the effect of thapsigargin on cytokine-induced degranulation of basophils is its effect on Ca2+ homeostasis in these cells. The effect of thapsigargin is duplicated by two other Ca2+-ATPase inhibitors (Table II), albeit with a lower efficacy. Moreover, the effect of thapsigargin is mimicked by the receptor agonist PAF, but again to a lower extent and in this case with a critical time dependence (Figure 7). The lower efficacy of PAF may either be due to the transient nature of the elevation in [Ca2+]i and/or to the transient Ca2+ store depletion. Moreover, we observed that the Ca2+ ionophore A23187 did not complement the cytokine signals in human basophils (Table II), indicating that an increase in [Ca2+]j per se is not compulsory for cytokine-induced histamine release. Due to the short incubation time of 15 min, also A23187 alone did not induce histamine release. The results obtained with thapsigargin in this study support the observations in RBL-2H3

50

Degranulation of human basophils by IL-3, IL-5 or GM-CSF

cells published by Lee and Oliver36. These authors suggested that inhibition of Ca2+ store refilling may be involved in activating Ca2+ influx after Fey R-l cross-linking, thus promoting degranulation of these cells. Our study in addition shows that emptying Ca2+ stores permits cytokine-induced histamine release from human basophils.

The pronounced effect of thapsigargin on the time course of histamine release induced by PMA (Figure 4) shows that activation of PKC alone is insufficient for the induction of efficient basophil degranulation, but requires additional signalling. The rapid degranulation response after PMA stimulation of thapsigargin-treated cells is in agreement with previous findings demonstrating optimal PMA effects within 10 min on PKC translocation or homotypic aggregation of human basophils32,37.

The study of cytokine-induced degranulation of human basophils has resulted in the current understanding that IL-3, IL-5 and GM-CSF only prime basophils for increased degranulation, but do not induce histamine release by themselves38"42. The potency of IL-3, IL-5 and GM-CSF to induce basophil degranulation after thapsigargin treatment is remarkable. This could also be demonstrated in the reverse set-up, i.e. first addition of picomolar concentrations of cytokines, followed by addition of thapsigargin (Figure 7). It indicates that the receptors for these cytokines on basophils are of high affinity, comprising functional dimers of a and ß chains43. Secondly, it further confirms previous reports that in humans IL-5 is active not only on eosinophils but also on basophils40,44,45. It might be interesting to see whether compounds like thapsigargin affect the phosphorylation pattern of the cytokine receptor, the tyrosine kinases concerned, the activation of the JAK involved (JAK2) or even the STAT protein46 or whether it exclusively complements the signal for degranulation by Ca2+ store activation, that can not be induced by cytokines. The effectiveness of picomolar concentrations of IL-3 after thapsigargin pretreatment questions the degranulative properties of IL-3 by itself that have been described with basophils from selected donors47, because in this study, as it is now clear, extremely high concentrations of IL-3 were used. We show here that the inability of IL-3, IL-5 and GM-CSF to induce histamine release is probably caused by the lack of effect on Ca2+ homeostasis in basophils. Recently, it has been demonstrated that some members of the C-C group of chemokines, such as RANTES, MCP-1 and MCP-3, can induce histamine release from human basophils48"51. Interestingly, these chemokines do induce changes in [Ca2+], by themselves51, which might explain the degranulative properties of these chemokines.

In allergic asthmatic disease, basophils are assumed to degranulate at sites of allergic inflammation, which might contribute to the local inflammatory process5 . The mechanisms determining degranulation of basophils in the

51

Chapter 2

tissues are unknown, but a role for residual allergen or complement fragments is unlikely53. Increased levels of mRNA encoding IL-3, IL-5 and GM-CSF, as well as their protein products have been demonstrated at sites of allergic late-phase responses54"57. IL-3, IL-5 and GM-CSF might be inducing histamine release of basophils at these tissue sites in conjunction with other stimuli that act on Ca2+

stores and induce Ca2+ release, such as PAF. In conclusion, the signal response cascade in human basophils at

suboptimal conditions for degranulation or after the addition of picomolar concentrations of IL-3, IL-5 or GM-CSF, is complemented by thapsigargin. The emptying of the Ca2+ stores leading to a Ca2+ rise might be an essential event for the degranulation of human basophils.

Acknowledgements

The authors thank G.M. Romijn-Tiele and F. Schotanus for the preparation of the buffy coats, B. Hooibrink for assistance in flow cytometric analysis and Dr. K.H. Krause, University Hospital Geneva, Switzerland for the gift of 2,5-di-(t-butyl)-1,4-benzohydroquinone.

Supported by the Netherlands Organization for Scientific Research NWO (project no. 900-512-163), by the Netherlands Asthma Foundation (project no 32.92.53) and by GlaxoWellcome BV, The Netherlands.

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2. Guo C-B, Liu MC, Galli SJ, Bochner BS, Kagey-Sobotka A, Lichtenstein LM. Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils. Am J RespirCell Mol Biol 1994;10:384-390.

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4. Hastie R, Chir B, Heroy JH, Levy DA. Basophil leukocytes and mast cells in human nasal secretions and scrapings studies by light microscope. Lab Invest 1979;40:554

5. Iliopoulos O, Baroody FM, Naclerio RM, Bochner BS, Kagey Sobotka A, Lichtenstein LM. Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils. J Immunol 1992;148:2223-2228.

6. Charlesworth EN, Hood AF, Soter NA, Kagey-Sobotka A, Norman PS, Lichtenstein LM. Cutaneous late-phase response to allergen. Mediator release and inflammatory cell infiltration. J Clin Invest 1989;83:1519-1526.

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Degranulation of human basophils by IL-3, IL-5 or GM-CSF

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